Project Summary/Abstract Antibiotic-resistant Gram-negative infections pose a serious threat to human health. The outer membrane of Gram-negative bacteria is a unique structure essential for survival; it also functions as a physical barrier to block entry of many classes of antibiotics, thereby rendering them ineffective. At present, colistin is the only antibiotic active against many resistant Gram-negative infections, but it has dose-limiting toxicity. We have discovered that the DNA gyrase inhibitor novobiocin and analogs with no gyrase activity potentiate the activity of colistin. Based on genetic, structural, and biochemical data, we propose that they do so by binding to and activating the inner membrane lipopolysaccharide transport complex. The inner membrane complex uses the energy of ATP hydrolysis by a cytoplasmic ATPase (LptB) to extract lipopolysaccharide from the inner membrane and move it onto a protein bridge that spans the periplasm to the outer membrane. We have shown that novobiocin binds at the interface between LptB and LptFG, the transmembrane components of the transporter. We have also shown that another component of the transporter, LptC, couples the energy of ATP hydrolysis to LPS extraction from the membrane. We propose experiments to better understand how LptC coordinates ATP hydrolysis with LPS extraction because this information is integral to understanding how novobiocin activates the machine. We also propose to develop new assays to quantify binding of novobiocin analogs and to monitor machine dynamics. We will apply these assays to characterize new synthetic analogs of novobiocin. Our objective is to identify compounds that strongly activate the transport machine while retaining gyrase activity. We will test compounds for synergy with colistin to test the hypothesis that strong activation of the transport machine is required for good synergy. This work may lead to development of safer colistin-based therapies to treat Gram-negative infections. In addition, the fundamental knowledge obtained about how this transporter works could enable discovery of other classes of compounds that interfere with its function.